Ultrastructural changes in the cell center during enterocyte differentiation in the mouse

Centrioles in the enterocytes of murine small intestine are located far away from the nuclei and close to the apical surface of cells (1-3 microns from the brush border). Centrioles never form a primary cilium and are not attached to the plasma membrane. Centrosomes (cell center) in enterocytes undergo subsequent involution in respect to the position of cell in the crypt-villus system. Five zones were studied separately: the bottom of the crypt (approx. 1/3 of the appendix), the upper part of the crypt (before its transition into the villus), the basal part, the middle and top of the villus. A centrosome in the crypt contains two centrioles (maternal and daughter) located close to each other. In 10 of 27 cells studied centrioles were replicating. A maternal centriole has 2-3 pericentriolar satellites and appendages. Several cytoplasmic microtubules run towards mother centrioles. Centrioles at the upper part of the crypt split at a distance up to 1.5 microns from each other. The average number of cytoplasmic microtubules and pericentriolar satellites decreases. At the basal part of the villus, centrioles split a distance up to 5 microns from each other; seldom microtubules may be found in a radius of 1 micron around centrioles and none of them is attached to centrioles or satellites. Starting from the middle of the villus, centrioles sometimes may have incomplete triplets of microtubules at the distal and (or) the proximal end. At the upper part of the villus there is only one centriole per cell, that was found in 10 of 50 cells studied on a complete series of 0.2 micron thick sections.     

Receptor editing occurs frequently during normal B cell development

Allelic exclusion is established in development through a feedback mechanism in which the assembled immunoglobulin (Ig) suppresses further V(D)J rearrangement. But Ig expression sometimes fails to prevent further rearrangement. In autoantibody transgenic mice, reactivity of immature B cells with autoantigen can induce receptor editing, in which allelic exclusion is transiently prevented or reversed through nested light chain gene rearrangement, often resulting in altered B cell receptor specificity. To determine the extent of receptor editing in a normal, non-Ig transgenic immune system, we took advantage of the fact that lambda light chain genes usually rearrange after kappa genes. This allowed us to analyze kappa loci in IgMlambda+ cells to determine how frequently in-frame kappa genes fail to suppress lambda gene rearrangements. To do this, we analyzed recombined VkappaJkappa genes inactivated by subsequent recombining sequence (RS) rearrangement. RS rearrangements delete portions of the kappa locus by a V(D)J recombinase-dependent mechanism, suggesting that they play a role in receptor editing. We show that RS recombination is frequently induced by, and inactivates, functionally rearranged kappa loci, as nearly half (47%) of the RS-inactivated VkappaJkappa joins were in-frame. These findings suggest that receptor editing occurs at a surprisingly high frequency in normal B cells.     

Platelet endothelial cell adhesion molecule-1 expression during mouse postimplantation development

The proteasome is an unusually large multisubunit proteolytic complex, consisting of a central catalytic machine (equivalent to the 20S proteasome) and two terminal regulatory subcomplexes, termed PA700 or PA28, that are attached to both ends of the central portion in opposite orientations to form the enzymatically active proteasome. Totally about 40 subunits with sizes of 20-110 kDa are assembled to form two types of the proteasomal complexes with the same catalytic core and different regulatory modules. To date, cDNAs or genes encoding almost all subunits of human and the budding yeast proteasomes have been isolated by molecular-biological techniques. In this minireview, I summarize briefly available information on the structure-function relationships of the proteasome acting as a protein death machinery.     

Differential effect of B lymphocyte-induced maturation protein (Blimp-1) expression on cell fate during B cell development

The B lymphocyte-induced maturation protein (Blimp-1) upregulates the expression of syndecan-1 and J chain and represses that of c-myc. We have transfected Blimp-1 into two sublines of the BCL1 B cell lymphoma that represent distinct stages of B cell development in secondary lymphoid tissues. After interleukin (IL)-2 and IL-5 stimulation, the BCL1 3B3 cells differentiate into centrocyte-like cells, whereas the BCL1 5B1b cells blast and appear to be blocked at the centroblast stage. This blasting effect and the increase in IgM secretion that follows it can be blocked by a dominant negative form of Blimp-1. At the same time, the ectopic expression of Blimp-1 in these partially activated cells induces an apoptotic response that also can be suppressed by the same dominant negative protein. A similar effect was noticed when Blimp-1 was expressed in the mature L10A and the immature WEHI-231 lines, indicating this may be a general effect at earlier stages of the B cell development, and distinct from the ability of Blimp-1 to induce maturation in late stages of differentiation. Truncation mutants indicate that the induction of the apoptotic response relies mainly on 69 amino acids within Blimp-1's proline-rich domain. We propose that Blimp-1 expression defines a checkpoint beyond which fully activated B cells proceed to the plasma cell stage, whereas immature and partially activated cells are eliminated at this point.     

Expression of an activated rasD gene changes cell fate decisions during Dictyostelium development

The authors describe iodinated contrast materials, which are commonly used in neuroradiological applications. The adverse reactions after their, administration are discussed. The indications for safer, non-ionic contrast media are presented. The protocol of prophylactic treatment of high risk patients is also formulated. We present the contrast material protocol used at our institution for neuroradiology.     

The binding of a subunit of the general polyadenylation factor cleavage-polyadenylation specificity factor (CPSF) to polyadenylation sites changes during B cell development

During the development of mouse B cells there is a regulated shift from the production of membrane (mb) to secretory-specific (sec) forms of immunoglobulin (Ig) mRNA. The mRNAs are produced from one gene that is alternatively processed at the 3' end. We have previously shown that there is an increase in polyadenylation efficiency accompanying the developmentally regulated shift to secretory-specific forms of Ig mRNA by DNA transfection experiments (1). When we look in vitro at nuclear extracts prepared from early/memory versus late stage/plasma B cells, we see cell stage-specific differences in the proteins which are crosslinked to poly(A) site-containing RNAs. Here we show that one of these proteins is the mouse homologue of 100 kDa subunit of Hela CPSF by immunoprecipitation and Western analysis of UV crosslinked material. The amount of 100 kDa protein and its mobility on two-dimensional gels do not change between the B cell stages. However, the binding of the 100 kDa polypeptide to poly(A) sites increases in the late stage/plasma cell lines relative to the binding seen in early/memory cell lines. The increased binding may reflect an increase in polyadenylation efficiency at the sec poly(A) site in plasma cells versus early/memory cells seen in vivo.     

Expression of mouse c-myb during embryonic development

Three members of the myb gene family, designated as A-myb, B-myb, and c-myb, have been described in many vertebrates. A large body of evidence indicates that the c-myb gene is essential for the development of most hematopoietic lineages. By contrast, the functions of A-myb and B-myb are less well understood. To further explore the relationship between the different myb family members we have compared the expression of A-myb and c-myb during mouse embryogenesis by Northern blotting and by in situ hybridization. In accordance with the important role of c-myb in the hematopoietic system, we detect high levels of c-myb expression in hematopoietic organs such as the fetal liver and the thymus. Surprisingly, we find that high levels of c-myb expression are not restricted to hematopoietic cells. We show that c-myb is strongly expressed in the neural retina and in epithelia of the respiratory tract. The side-by side analysis of c-myb and A-myb expression clearly shows that both genes are expressed in different, but overlapping sets of tissues. Our results suggest that the function of c-myb may not be restricted to hematopoietic cells.     

Apoptosis during B lymphopoiesis in mouse bone marrow

To evaluate the magnitude of cell death and the critical stages at which it occurs during B lymphopoiesis in mouse bone marrow (BM), we have examined the kinetics of apoptosis at defined stages of B cell differentiation. FACS-sorted B220+ BM cells exhibited a low incidence of morphologically apoptotic cells by electron microscopy. In freshly prepared BM suspensions, the incidence of hypodiploid cells detected by multiparameter flow cytometry was greater among large dividing B220+ surface IgM- (sIgM-) precursor B cells and sIgM(low) immature B lymphocytes than among terminal deoxynucleotidyl transferase+ (TdT+) pro-B cells, small nondividing B220+ sIgM- precursors, and surface IgD+ mature B lymphocytes. During short-term culture, apoptotic cells, identified by both DNA content and in situ DNA strand break labeling, increased linearly with time without macrophage ingestion, providing an assay for the rate of entry into apoptosis. B220+ B lineage cells accumulated in apoptosis more rapidly than cells of other lineages. The apoptotic rate was greater among B220+ sIgM- precursor cells than sIgM+ B cells, and was highest among B220+ mu- pro-B cells. Coculture with stromal cells reduced the apoptotic rate of B220+ sIgM- precursors to a greater extent than that of sIgM+ B lymphocytes. The results lead to estimates of the actual number of B lineage cells undergoing apoptosis per unit time in successive differentiation compartments. The findings indicate that, although influenced by local microenvironmental factors, apoptotic cell death occurs most markedly at two developmental stages associated with Ig heavy chain gene rearrangement and Ag receptor expression, respectively.     

Biochemical and functional changes during the bovine fetal thyroid development

Despite improvements in critical care medicine and the development and aggressive use of potent broad-spectrum anti-microbial agents, mortality due to severe sepsis has not changed during the recent years and still comes to 35% to 45%. For quite a long time our understanding of the pathophysiology of sepsis was mainly focused on endotoxin and proinflammatory cytokines like tumor necrosis factor or interleukin-1. Now it is generally accepted that many signs and symptoms of sepsis are not directly mediated by cytokines but are transmitted through other mediator systems. The coagulation system comes into play especially when the septic process progresses to malperfusion and organ failure. Antithrombin III is an important inhibitor of the intrinsic, extrinsic and common pathway of coagulation. Recently, evidence has been accumulating that there is an additional anti-inflammatory potential of the drug. Currently there are several clinical trials ongoing to investigate whether this effect is of clinical relevance in the treatment of patients with severe sepsis.     

V-D-J rearrangements at the T cell receptor delta locus in mouse thymocytes of the alpha beta lineage

The T cell receptor (TCR) delta locus lies within the TCR alpha locus and is excised from the chromosome by V alpha-J alpha rearrangement. We show here that delta sequences persist in a large fraction of the DNA from mature CD4+CD8- alpha beta+ mouse thymocytes. Virtually all delta loci in these cells are rearranged and present in extrachromosomal DNA. In immature alpha beta lineage thymocytes (CD3-/loCD4+CD8+) and in CD4+CD8- alpha beta+ thymocytes expressing a transgene-encoded alpha beta receptor, rearranged delta genes are present both in chromosomal and extrachromosomal DNA. Thus, contrary to earlier proposals, commitment to the alpha beta lineage does not require recombinational silencing of the delta locus or its deletion by a site-specific mechanism prior to V alpha-J alpha rearrangement.     

Programmed cell death in the development of the mouse external auditory canal

Programmed cell death (PCD) is an essential event for development. The purpose of this work was to ascertain how PCD, in vivo designated apoptosis, is involved in the development of the external auditory canal. We performed a time sequence study of the distribution of apoptosis during the development of external auditory canal (EAC) of the mouse. ICR mice ranging in age from embryonic day 11.5 (E11.5) to 12 days after birth (DAB) were used in the present study. A part of each head including both ears was removed and was processed according to its purpose. Light and electron microscopy for morphological studies and TUNEL method (Gavrieli et al. [1992] J Cell Biol., 119:493-501) for histochemical studies were used. On E11.5, distinct TUNEL-positive staining occurred in the branchial arch. Between E15.5 and 1DAB, TUNEL-positive cells were observed throughout the EAC and the number of these cells decreased with age. On E15.5 and E16.5, numerous TUNEL-positive cells were observed in a cavity remained in the epithelial plate. Transmission electron microscopy revealed that these cells had the features of apoptosis. From 3-12 DAB, no apoptosis was observed in the EAC except for the terminal differentiation of the skin of the EAC. Apoptosis was not observed during recanalization of the EAC, but occurred during the formation of the epithelial plate. The investigation established that PCD is involved in the formation of the epithelial plate, whereas only cornification of the epithelium of the EAC is associated with recanalization.     

Haematological changes during development of acute laminitis hypertension

Experimental carbohydrate overload of 15 horses resulted in consistent haematological changes during development of laminitis hypertension. Significant alterations in packed cell volume, leucocyte differential count, serum glucose levels and protein values occurred before onset of Obel grade 3 lameness and hyperkinetic circulatory state. Blood platelets were significantly decreased 8 h after the onset of severe lameness. Findings in this study were indicative of haemoconcentration due to compartmental fluid shifts and leucocytic stress response consistent with increased circulating adrenoglucocorticoids and/or catecholamines.     

Hemispheric changes in information processing during development.

Hemispheric processing of visually presented words and pictures was examined in 3 groups of Ss with mean ages of 8 yrs 8 mo, 12 yrs 3 mo, and 27 yrs 9 mo (154 Ss). Pictorial or symbolic stimuli were presented singly to either the right or left visual hemifield. Ss had to decide whether the 1st stimulus in a pair matched the 2nd stimulus. The major results were that (a) age groups differed in the strength of lateral differences, and (b) for all age groups, the right hemisphere was significantly faster in processing unmatched stimuli. Results suggest that lateral specialization of the left hemisphere is not complete until adolescence and that over the age range tested, the left hemisphere becomes progressively more specialized for the processing of matched data. (52 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Biochemical changes in cottonseed during development and maturity

A simple semiquantitative slide test to assess the phagocytic activity of periphera polymorphonuclear leukocytes is described. Leukocytes are isolated from one drop of blood and latex particles are offered for phagocytosis. Cellular capacities and serum opsonins can be assessed separately. In the presence of pooled adult serum no defect of phagocytosis was seen in preterm and full-term newborns nor in pediatric patients with infectious or other diseases. In 4 of 7 premature newborns a definite deficiency of serum opsonins was demonstrated. In in vitro studies complement (plus possibly other heat-labile factors) promoted phagocytosis. Phagocytic rates were depressed by endotoxin and phenylbutazone.     

Tropomyosin in preimplantation mouse development: identification, expression, and organization during cell division and polarization

Tropomyosin is an actin-binding cytoskeletal protein which has been extensively characterized in a variety of cell types and tissues, with the exception of very early developmental stages during which cellular polarization first occurs. We have identified five polypeptides in mouse preimplantation conceptuses which show many of the characteristics of tropomyosin. They form the major portion of the heat-stable cytoskeletal protein fraction of blastomeres and have the characteristic isoelectric and SDS-PAGE migration characteristics on 1-D and 2-D gels. All five polypeptides were synthesized in late 2- and 4-cell, and all 8-cell stages, with three of the five polypeptides showing lower synthetic levels in fertilized eggs and early 2-cell conceptuses. These heat-stable proteins showed specific differences from proteins isolated from mouse 3T3 fibroblasts by the same method, namely higher Mr isoforms were not represented, also some of the isoforms can be labeled by incorporation of [14C]proline. The cellular distribution of tropomyosin in early stage conceptuses was examined using monoclonal and affinity-purified polyclonal antibodies. Tropomyosin becomes associated both with the blastomere cortex postfertilization and with the cleavage furrow during cytokinesis. The interphase cortical association is uniform until the 8-cell stage, when tropomyosin becomes associated with the developing apical pole and is excluded from the basolateral cortex. This polar localization is inherited along with the pole at the 8- to 16-cell division, but experiments in which cell division is artificially prolonged show that tropomyosin localization does not represent a permanent marking of the pole. We conclude that the early mouse conceptus contains a unique and specific set of tropomyosins which respond to polarizing signals.     

Characterization of mutations at the mouse phenylalanine hydroxylase locus

By using a three-dimensional computed tomography (CT) scanner, we compared the anatomic features of the pelvis of three fetuses of same gestational age, one with a normal pelvis representing the reference model, one with classic bladder exstrophy, and one with cloacal exstrophy. The tomography slices were selected at the same levels for each case. Three angles expressing external opening of the pelvis were defined. Comparing normal and abnormal pelvises allowed definition of three criteria for the correction of the malformation: (a) the sum of the differential angles gives the amplitude of the correction needed; (b) a supraacetabular osteotomy appears to allow best closure of the pelvic ring; (c) only three slices of a CT scan are needed, which cannot be harmful, especially for neonates. Therefore, we believe that a CT scan of the pelvis should be performed whenever an osteotomy is planned in the surgical reconstruction of bladder and cloacal exstrophy.